. Primary mitochondrial respiratory chain (RC) diseases and congenital disorders of glycosylation (CDG) are collectively common metabolic diseases that cause an overlapping spectrum of disrupted brain development and multi-systemic disease. Much remains to be discovered about the nature and regulation of the mitochondrial glycoproteome and its potential relevance to inherited disorders of RC function or glycosylation. Our preliminary data suggest that: [1] Mitochondria from diverse species have unique N-linked glycome modifications assessed by MALDI-TOF mass spectrometry (MS); [2] Several RC proteins are N-glycosylated; and [3] Patient fibroblast cell lines (FCL), C. elegans, and mouse strains lacking the N-glycanase NGLY1 have mitochondrial depletion, RC dysfunction, and increased oxidative stress. The overall goal of this proposal is to characterize the mitochondrial RC glycoproteome and its regulation, and clarify its relevance to inherited disorders of RC function and NGLY1 activity. We hypothesize that glycosylation and deglycosylation of mitochondrial glycoproteins, including subunits of RC complexes, are essential for maintenance of normal mitochondrial function. The Specific Aim of this proposal is to characterize the N-linked glycoproteome of the RC and the ways it is altered by RC disease. In silico predictions that specific RC proteins are N-glycosylated will be tested by lectin reactivity and PNGaseF, endoglycosidase H and F sensitivity of blue-native gel separated RC complexes. The glycoproteins and their glycosylation sites as well as the glycan structure will be identified by mass spectrometry. Effects of RC inhibition due to either genetic disease or pharmacologic inhibition on glycoprotein modifications of the mitochondrial RC will be characterized in patient FCLs from RC disease and NGLY1 deficient patients, as well as from mouse NGLY1 deficient MEF models. These studies will identify N-glycosylated RC proteins and elucidate how they are impacted by RC disease.